Abstract
BackgroundLoss-of-function mutations in the dysferlin gene (DYSF) result in a family of muscle disorders known collectively as the dysferlinopathies. Dysferlin-deficient muscle is characterized by inflammatory foci and macrophage infiltration with subsequent decline in muscle function. Whereas macrophages function to remove necrotic tissue in acute injury, their prevalence in chronic myopathy is thought to inhibit resolution of muscle regeneration. Two major classes of macrophages, classical (M1) and alternative (M2a), play distinct roles during the acute injury process. However, their individual roles in chronic myopathy remain unclear and were explored in this study.MethodsTo test the roles of the two macrophage phenotypes on regeneration in dysferlin-deficient muscle, we developed an in vitro co-culture model of macrophages and muscle cells. We assayed the co-cultures using ELISA and cytokine arrays to identify secreted factors and performed transcriptome analysis of molecular networks induced in the myoblasts.ResultsDysferlin-deficient muscle contained an excess of M1 macrophage markers, compared with WT, and regenerated poorly in response to toxin injury. Co-culturing macrophages with muscle cells showed that M1 macrophages inhibit muscle regeneration whereas M2a macrophages promote it, especially in dysferlin-deficient muscle cells. Examination of soluble factors released in the co-cultures and transcriptome analysis implicated two soluble factors in mediating the effects: IL-1β and IL-4, which during acute injury are secreted from M1 and M2a macrophages, respectively. To test the roles of these two factors in dysferlin-deficient muscle, myoblasts were treated with IL-4, which improved muscle differentiation, or IL-1β, which inhibited it. Importantly, blockade of IL-1β signaling significantly improved differentiation of dysferlin-deficient cells.ConclusionsWe propose that the inhibitory effects of M1 macrophages on myogenesis are mediated by IL-1β signals and suppression of the M1-mediated immune response may improve muscle regeneration in dysferlin deficiency. Our studies identify a potential therapeutic approach to promote muscle regeneration in dystrophic muscle.
Highlights
Loss-of-function mutations in the dysferlin gene (DYSF) result in a family of muscle disorders known collectively as the dysferlinopathies
Inhibition of IL-1β ameliorates muscle differentiation in A/J myoblasts Having shown that IL-4 activity alone is not sufficient to rescue muscle differentiation in A/J muscle cells, we focused on factors released from M1 macrophages that inhibit muscle differentiation
We did not test for neutrophil recruitment, our studies suggest that the M1-mediated response to injury is attenuated in Bla/J muscle, which we attribute to chronic M1 macrophage infiltration and upregulation of M1-derived IL-1β and Nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) in the absence of dysferlin
Summary
Loss-of-function mutations in the dysferlin gene (DYSF) result in a family of muscle disorders known collectively as the dysferlinopathies. The most common manifestations include limb-girdle muscular dystrophy 2B (LGMD2B), an autosomal recessive myopathy marked by proximal muscle weakness, with an onset in the late teens ([2, 3], reviewed in [4]) and Myoshi myopathy, characterized by a progressive muscle wasting involving distal muscles ([5] reviewed in [4]) These myopathies feature chronic regeneration and fibrosis [6], a selective loss of type 2 muscle fibers and a moderate degree of inflammation surrounding the necrotic fibers [7], the severity of pathology is variable. The primary infiltrating cells in dystrophic muscle are macrophages, which have been studied more extensively in the mdx mouse model of Duchenne muscular dystrophy [9]
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